Difference between revisions of "Amplex UltraRed"

Revision as of 21:35, 6 February 2016

high-resolution terminology - matching measurements at high-resolution

Amplex UltraRed

Description

Amplex red (AmR) is used as an extrinsic fluorophore for measurement of hydrogen peroxide production (ROS) by cells or mitochondrial preparations. The reaction of H₂O₂ and AmR is catalyzed by horseradish peroxidase to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm). The change of emitted fluorescence intensity is directly proportional to the concentration of H₂O₂ added, whereby the H₂O₂ is consumed.

Abbreviation: AmR

Reference: Mohanty 1997 J Immunol Methods, Zhou 1997 Anal Biochem, Mishin 2010 Free Radical Biol Med, Towne 2004 Anal Biochem, Krumschnabel 2015 Methods Mol Biol

MitoPedia methods: Fluorometry

Media

See Krumschnabel 2015 Methods Mol Biol, Tretter 2012 Free Radic Biol Med, Komary 2010 Biochim Biophys Acta.

Media with high antioxidant activity compete with HRP and partially consume H₂O₂ before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This was shown by comparing

the sensitivity as determined by H₂O₂ additions to different media containing HRP and Amplex (R) UltraRed;
the sensitivity as determined by addition of the actual fluorophore resorufin to the same media (signal change per added resorufin); the sensitivity is signal change per added H₂O₂ or resorufin.

The sensitivity, as determined by addition of H₂O₂, is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the sensitivity with respect to resorufin.

A chemical background drift, which is always observed for the Amplex /HRP system, is independent of the H₂O₂ sensitivity versus the same (absolute) drift expressed as (calibrated) "apparent H2O2 production" will be higher in a medium with low H2O2 sensitivity as compared to a medium with high H2O2 sensitivity.

As expected some traditional KCl based media, similar to those used in ... showed a far higher sensitivity against H2O2 addition than MiR05, while some experiments indicated a lower respiration.

Different brands

Trade Mark	Manufacturer/ Distributor	price [€/mg]	product id
Amplex Red	Life Technologies	37.4	A12222
Amplex Ultra Red	Life Technologies	48.8	A36006
Ampliflu Red	Sigma	18.5	90101
Quanta Red	Thermo scientific		15150

Citation	Amplex		HRP			pH	Limit of detection
	stock	final	unit definition	stock	final
	mM	µM		U/ml	U/ml
BIOTEK	10	50	pyrogallol	10	0.1	7.4	4 nM (absorption 300 nm)
Life Technologies	10	50		10	0.1	6 to 7.5	<80 nM
Towne 2004		160			0.41	7.5 to 8.5	100 nM
Zhou1997		3 ?			0.3 to 1		50 nM (10 nM optimal)
Mohanty 1997	100	10 to 100			1		18 nM
Komary 2010		1			2.5

Applications with O2k-Fluorometry

Source: Life Technologies (former Invitrogen) A36006

Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. »Bioblast pdf«
Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. »Bioblast pdf«

O2k-Demo experiments

@@ Line 9: / Line 9: @@
 {{MitoPedia topics}}
-== Signal and output ==
+== Media ==
-# Signal: The [[O2k-Fluo LED2-Module]] is operated through the  amperometric (Amp)-Channel of the O2k, with electric current (ampere  [Amp]) as the primary signal.
-# Output: The focus of the output with Amplex red is on '''[[O2k signals and output| Type B]]''': Flow, flux, rate.
+See [[Krumschnabel 2015 Methods Mol Biol]], [[Tretter 2012 Free Radic Biol Med]], [[Komary 2010 Biochim Biophys Acta]].
+Media with high antioxidant activity compete with HRP and partially consume H<sub>2</sub>O<sub>2</sub> before it can react with Amplex (R) (Ultra)Red  to form the active fluorophore resorufin. This was shown by comparing
+* the sensitivity as determined by H<sub>2</sub>O<sub>2</sub> additions to different media containing HRP and Amplex (R) UltraRed;
+* the sensitivity as determined by addition of the actual fluorophore resorufin to the same media (signal change per added resorufin); the sensitivity is signal change per added H<sub>2</sub>O<sub>2</sub> or resorufin.
+The sensitivity, as determined by addition of H<sub>2</sub>O<sub>2</sub>, is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the sensitivity with respect to resorufin.
+A chemical background drift, which is always observed for the Amplex /HRP system, is independent of the H<sub>2</sub>O<sub>2</sub> sensitivity versus the same (absolute) drift expressed as (calibrated)  "apparent H2O2 production" will be higher in a medium with low H2O2  sensitivity as compared to a medium with high H2O2 sensitivity.
+As  expected some traditional KCl based media, similar to those used in   ... showed a far higher sensitivity against H2O2 addition than MiR05, while some experiments indicated a lower respiration.